家蚕丝氨酸蛋白酶抑制剂5的多克隆抗体制备及组织表达分析  

Preparation of Polyclonal Antibody and Tissues Expression Analysis of Bombyx mori Serine Protease Inhibitor 5

作　　者：李国胜[1,2] 王彦云[1,2] 王明慧[1,2] 徐开遵[1,2] 陈玉华[1,2,3] 沈卫德[1,2,3] 许雅香[1,2,3] 

机构地区：[1]现代丝绸国家工程实验室,江苏苏州215123 [2]苏州大学基础医学与生物科学学院,江苏苏州215123 [3]苏州大学蚕桑研究所,江苏苏州215123

出　　处：《蚕业科学》2013年第2期261-265,共5页ACTA SERICOLOGICA SINICA

基　　金：现代农业产业技术体系专项(No.CARS-22);江苏高校优势学科建设工程项目

摘　　要：为探讨家蚕丝氨酸蛋白酶抑制剂5(Serpin-5)的功能,制备其多克隆抗体并分析该蛋白质在家蚕组织中的表达特征。克隆获得去除信号肽的家蚕serpin-5基因,该基因大小为1 140 bp,编码379个氨基酸残基,预测蛋白质分子质量42.6 kD,等电点6.02。构建用于原核表达的pET28a-serpin-5载体,并在E.coli BL21中进行重组蛋白的诱导表达。经SDS-PAGE分析表明重组家蚕Serpin-5蛋白多以包涵体的形式存在,纯化后的融合蛋白的分子质量与预测值相符。以此为抗原免疫昆明小鼠,Western blot检测制备的多克隆抗体具有较高的特异性,ELISA检测该多抗对免疫抗原的效价达到1∶24 000。在制备高效价抗体的基础上,通过Western blot检测家蚕Serpin-5蛋白在5龄第3天幼虫精巢、卵巢中的表达量较高,中肠次之,在丝腺、马氏管、脂肪体中的表达量较低。推测家蚕Serpin-5蛋白在生殖腺中的高表达可能与家蚕的生殖功能相关。In order to understand function of serine protease inhibitor 5(Serpin-5) in silkworm(Bombyx mori),we prepared its polyclonal antibody and analyzed its expression pattern in various silkworm tissues.We first cloned and obtained the Bombyx mori serpin-5 gene without signal peptide.This gene is 1 140 bp long and encodes 379 amino acid residues with predicted protein molecular weight around 42.6 kD and isoelectric point of 6.02.Then we constructed vector pET28a-serpin-5 for prokaryotic expression and carried out induced expression of recombinant protein in E.coli BL21.SDS-PAGE analysis showed that the recombinant protein Serpin-5 was expressed as inclusion body.The purified fusion protein had molecular weight consistent with the predicted value.This protein was used as antigen to immune Kunming mice.Western blot analysis displayed that the prepared polyclonal antibody had high specificity.ELISA assay indicated that the anti-immune antigen titer of this polyclonal antibody reached 1 ∶24 000.Based on preparation of high titer antibody,Western blotting revealed that Serpin-5 protein had high expression levels in testis and ovary of day 3 silkworm larvae of the 5th instar,followed by midgut,silk gland,Malpighian tubule and fat body.The high expression of Serpin-5 in silkworm gonads showed that this protein is potentially involved in silkworm reproductive process.

关 键 词：家蚕 丝氨酸蛋白酶抑制剂 原核表达 多克隆抗体 组织表达 

分 类 号：S881.2[农业科学—特种经济动物饲养] Q78[农业科学—畜牧兽医]